Identification and characterization of the major Huperzine A metabolite in rat blood

Huperzine A (Hup A) is under investigation as a treatment of Alzheimer's disease because of its properties of reversible and specific AChE inhibition. It has additional interesting pharmacological effects such as the protection of primary neuronal cells isolated from embryonic rat brains from glutamate-induced toxicity. We have isolated a new compound which has similar absorbance characteristics as Hup A from blood of rats administered Hup A. Monitoring the effluent from reversed-phase high-performance liquid chromatography (RP-HPLC) of blood collected 60 min after Hup A treatment at an absorbance of 308 nm (λmax for Hup A), yielded a peak height and area for this compound that was ∼1.4-fold the initial Hup A peak. The compound was isolated from RP-HPLC fractions from blood and liver for analysis by mass spectrometry and nuclear magnetic resonance (NMR). The compound gave an (M+H)⁺ ion with m/z 259 in positive ion mode, yielding a molecular weight (MW) of 258. If derived from Hup A (MW 242), the change in MW indicates a mass gain of 16. This would be consistent with the addition of a single oxygen or a hydroxylation. To determine the location of the modification, it was examined by ¹H NMR, and it was found that the added mass was due to a single epoxidation yielding 13,14-epoxy Hup-A.